Mitochondrial disease occurs about 1 in 5000 births. The enzyme most commonly affected is mitochondrial Complex I (NADH:ubiquinone oxidoreductase), the entry point of the electron transport chain. It oxidizes NADH, reduces ubiquinone, and couples the free energy from those reactions to the translocation of protons across the membrane, which are used to drive the synthesis of ATP. In humans, 7 of the 45 subunits of Complex I are encoded by mitochondrial genes. Advances in sequencing of genomes and exosomes has greatly increased the number of gene variants found in mitochondrial disease, but it has been difficult to determine if these alleles are causative for disease. In the case of mitochondrial genes, it is complicated both by heteroplasmy, in which both wild type and mutant alleles can co-exist in the same cell, and by the various haplotypes of mitochondrial DNA that can influence the effects of a mutation. A bacterial model system allows individual mutations to be examined in a consistent background. Recent structural studies have demonstrated that the 14 core subunits of Complex I from mitochondria are nearly identical to the 14-subunit bacterial enzyme. The goal of this proposal is to characterize about 24 clinical mutations in a bacterial version of Complex I, to provide insight into their effects on assembly and function, and to improve disease diagnosis. These mutations are associated with disease, but have not been shown to be causative. In the first aim, mutations that map to interfaces of ND2, ND4, and ND5 subunits at the distal end the membrane arm will be analyzed. It will be determined whether the mutations impair enzyme activity, or prevent the binding of one of the partner subunits, and how that impacts the assembly process. In the second aim, mutations that map to the integrated pair of ND6-ND4L will be analyzed. It will be determined whether these mutations disrupt mutual interactions, or interactions with neighboring subunits, such as ND2 and ND5, and how that affects assembly of Complex I. In the third aim, mutations will be modeled that map to ND1-ND3, at the interface between the membrane subunits and the peripheral arm. Effects on enzyme activity and assembly will be examined. Assembly will be verified by native gel electrophoresis and western blotting. The results will provide insights into the assembly pathways of Complex I, and the nature of disruptions by clinically-identified mutations in the core subunits. Results will provide guidance in the diagnosis of patients with currently-known, and newly-identified mutations.